Bifidobacterium growth promotant

ABSTRACT

This invention relates to a bifidobacterium growth promotant comprising gluconic acid, a nontoxic salt thereof and/or glucono-δ-lactone as an active ingredient. 
     The bifidobacterium growth promotant of this invention has selective bifidobacterial growth promoting-activity and, at the same time, inhibits growth of deleterious bacteria. Moreover, its rate of digestion and absorption in the upper alimentary tract is so low that the promotant has very satisfactory characteristics as a bifidus factor. Therefore, the bifidobacterium growth promotant of this invention can be used per se or as an additive for various foods and drink to provide functional foods and drinks, thus being of great value from the standpoint of health improvement.

TECHNICAL FIELD

This invention relates to a growth promotant having a selectivebifidobacterium growth promoting-effect and the use thereof in variousapplications.

BACKGROUND ART

Microorganisms of the genus Bifidobacterium, alias bifidobacteria, areconstituents of the intestinal flora in mammals inclusive of man and itis known that these bacteria per se are not pathogenic but ratherantagonizing pathogenic intestinal bacteria in respect of, for example,lactic acid production and nutrient requirement, thus interfering withproliferation of the pathogenic bacteria in the intestinal canal. It isalso known that a breast-fed infant in whom these bacteria are presentin the form of pure cultures is less susceptible to enteral infectionthan a bottle-fed infant in whom they are only sparsely present. Basedon the above and other findings, many progresses have been made in theresearch concerning bifidobacteria of late and as more light is cast onthe immuno-potentiating effect, prophylactic effect on microbialsubstitution, and inhibitory effect on carcinogenic substances of thesebacteria, their clinical application has been attempted.

Since bifidobacteria must be encouraged to multiply in the intestine inorder that their variegated actions may be exploited, much research intosubstances that could promote growth of bifidobacteria (hereinafterreferred to as bifidus factors) is going on with great enthusiasm.

The generally acknowledged requisites of any bifidus factor are that itis not digested and absorbed in the upper digestive tract but reachesthe ileum and large intestine and that it is efficiently assimilated bybifidobacteria and hardly utilized by other bacteria. Several substancesin the name of bifidus factors have heretofore been proposed and, aboveall else, incorporation of oligosaccharides in various kinds offoodstuffs is broadly practiced.

However, from the standpoint of said requisites, the oligosaccharides sofar reported are not impeccable bifidus factors. Fructooligosaccharides,for instance, have the disadvantage that they are decomposed by gastricacid and partly absorbed from the upper digestive tract.Isomaltooligosaccharides, too, are hydrolyzed by small intestine mucosalenzymes and absorbed there, thus making it mandatory to ingest them inmassive doses. Galactooligosaccharides are relatively resistant to acidand heat and not easily digested and absorbed in the upper digestivetract but have the drawback, process-wise, that their yields are low andthe costs of production are high. Furthermore, since all of them aresugars, they act as sweeteners when applied to food so that thedisadvantage of sweetness alone being emphasized in the food isunavoidable. In addition, the utilization selectivity of bacteria forthese saccharides is low. That is to say they are utilized not only bybifidobacteria but also by other intestinal bacteria, thus presentingthe problem that a massive intake is necessary for achieving sufficientefficacy.

Having been developed with a view to overcoming the above-mentioneddisadvantages of the prior art, this invention has for its object toprovide a bifidus factor which is not digested and absorbed in the upperdigestive tract, promotes growth of bifidobacteria remarkably, and doesso with high selectivity.

DISCLOSURE OF INVENTION

This invention, made to accomplish the above object, is directedessentially to the incorporation of gluconic acid, a nontoxic saltthereof and/or glucono-δ-lactone as an active substance.

The inventors of this invention did intensive research about bifidusfactors and discovered that gluconic acid, a nontoxic salt thereofand/or glucono-δ-lactone has activity to promote growth ofbifidobacteria with good selectivity and, based on this new finding, didfurther research. This invention has been developed on the basis of theabove research. Thus, the inventors' research into the bacterialutilization of gluconic acid revealed that said substance is efficientlyutilized by Bifidobacterium adolescentis, among bifidobacteria, which isreportedly predominant in the intestinal tract of the adult human and isnot utilized by Clostridium perfringens, which is a causative agent ofpuerperal fever, appendicitis, enteritis and food poisoning, but ratherinhibits its growth. It was also elucidated that gluconic acid (salt) isnot utilized by bacteria of the genus Bacteroides which are said to bedominant in the intestine.

It is described in Bergy's Manual of Systematic Bacteriology, 8th ed.,the Williams and Wilkinson, that gluconic acid (salt) is utilized byBifidobacterium adolescentis in vitro. Whereas, as mentioned above, animportant requisite of a bifidus factor is that it is not digested andabsorbed in the upper digestive tract but reaches the large intestine,it is known that organic acids in general are absorbed in the smallintestine, suggesting that gluconic acid would also be absorbed in thesmall intestine. Moreover, since gluconic acid resembles glucose inchemical structure, it has been believed that like glucose it isabsorbed in the small intestine. However, as described hereinafter, anexperiment about the small intestinal absorption of gluconic acid underthe conditions providing for 100% absorption of glucose in theintestinal loop assay revealed surprisingly that a major proportion ofgluconic acid is not absorbed in the small intestine but reaches thelarge intestine. It is a new finding that an organic acid has activityto promote the growth of bifidobacteria. In addition, the inventors ofthis invention should be credited with the discovery that gluconic acidinhibits growth of Clostridium perfringens which is termed "harmfulbacteria." Thus, gluconic acid has activity to promote selective growthof bifidobacteria.

Among species of bifidobacteria, it is known that not only saidBifidobacterium adolescentis but also Bifidobacterium pseudocatenulatum,Bifidobacterium catenulatum, etc. are capable of utilizing gluconicacid. Therefore, gluconic acid, a nontoxic salt thereof andglucono-δ-lactone are able to promote growth of these bacteria.

Since gluconic acid has a mild sour taste, it is used as a foodacidulant, and because its calcium salt is readily soluble in water, ithas been used as a calcium or supplement for food or medicinal use.Glucono-δ-lactone, when dissolved in water, converts itself gradually togluconate and is in use as a coagulant in the production of soybeancurds and baking powder of bread.

The nontoxic salt of gluconic acid for use in this invention is notrestricted, inasmuch as it is physiologically harmless and efficientlyutilized by bifidobacteria. Thus, alkali metal salts, e.g. sodium salt,potassium salt, etc., calcium salt, magnesium salt, zinc salt, coppersalt, etc. can be mentioned as preferred examples.

Gluconic acid, a salt thereof and/or glucono-δ-lactone, which asaforesaid has activity to promote growth of bifidobacteria, can be usedas a bifidobacterium growth promotant, a Clostridium growth inhibitor,an intestinal function controlling agent, an intestinal putrefactivefermentation inhibitor, a prophylactic and therapeutic agent fordiarrhea, a stool deodorant, a stool property improving agent or ananticonstipation drug in man and animals, and even as an animal growthpromotant, among other uses.

A variety of compositions for diverse uses, inclusive of those for useas a bifidobacterium growth promotant in accordance with this inventioncan be provided in a variety of dosage or application forms such aspowders, granules, tablets, etc. each containing gluconic acid, anontoxic salt thereof and/or glucono-δ-lactone, optionally incombination with other bifidobacterium growth promotant. Depending onspecific uses and objectives, such compositions can be provided inliquid forms.

Furthermore, the compositions for various uses, inclusive of those foruse as a bifidobacterium growth promotant, according to this inventionmay contain a variety of conventional additives with advantage. Includedamong such additives are dietary fibers such as apple pulp fiber, cornfiber, alginic acid, carrot powder, pectin, seaweed polysaccharide,carboxymethylcellulose, etc.; excipients such as lactose, starch, etc.;sweeteners such as sucrose, maltose, fructose, sorbitol, mannitol,stevioside, aspartame, etc.; nutritional supplements such as vitamins,minerals, skim milk, meat extract, etc.; flavors; binders such as gumarabic powder, polyvinylpyrrolidone, hydroxypropylcellulose, etc.; andlubricants such as magnesium stearate, calcium stearate, talc, etc.;among other additives. These additives can be selectively employed.

The bifidobacterium growth promotant or other compositions of thisinvention can be administered to man and animals either in the form ofgluconic acid, a nontoxic salt thereof and/or glucono-δ-lactone as suchor in the form of a suitable preparation. As a further alternative, theycan be added to food for human consumption or feed for animals.

To be specific, they can be added to fermented milk, soft drinks,sherbets, candies, jellies and other confectionaries, soybean curds,meat and fish cakes and sausages, and other foods (e.g. various pickles,dressings, etc.). Also, for the purpose of a stool deodorant, saidcompositions can be added to diets for the aged or to hospital rations.The powders and granules can be added to fish flour, instant soup,instant "miso" soup, etc. Moreover, for the prophylaxis and therapy ofdiarrhea, attenuation of fecal odor, or growth promotant, it can be usedas feed additive for cattle, swine, poultry and other domestic animalsand pet animals such as the dog and cat. As a further mode of use,gluconic acid, a nontoxic salt thereof and/or glucono-δ-lactone can beadded to drinking water for man and animals.

Furthermore, while gluconic acid, a nontoxic salt thereof and/orglucono-δ-lactone has already been in use as an acidulant, calciumsupplement (calcium gluconate), coagulant, or baking powder, it can beused as a bifidus factor additionally having the functions of suchagents.

The preferred concentration in food or feed is 0.1-10 weight %. Whilethe nontoxicity of gluconic acid is well known, the daily total intakecan be about 0.1 g/kg for adults and about 0.05 g/kg for infants andchildren as rule of thumb.

The various effects, inclusive of the effect of promoting growth ofbifidobacteria, of this invention can also be expected from the use ofthose substances which are hydrolyzed to liberate gluconic acid inwater, such as various esters of gluconic acid (e.g. alkyl esters suchas methyl ester, ethyl ester, etc.), glucono-γ-lactone, etc. or evenfrom the use of D-galactonic acid, D-galacto-1,4-lactone and so on.

The food and food materials containing gluconic acid, a nontoxic saltthereof and/or glucono-δ-lactone, which has a selective growth promotingeffect on bifidobacteria, are of use as functional foods and foodmaterials.

With regard to means for containing gluconic acid, a nontoxic saltthereof or glucono-δ-lactone in a substrate food or food material, adirect addition method and a method which comprises causing a componentof a food or food material to be chemically or biochemically convertedto gulconic acid can be utilized.

When the former method is employed, a suitable member can be selectedfrom among gluconic acid, a nontoxic salt thereof and glucono-δ-lactoneaccording to the specific objective. Thus, for example, where theobjective is addition of a sour taste, gluconic acid and/orglucono-δ-lactone or an equivalent thereof can be added. For the purposeof addition of a salty taste or improving the taste, the addition ofsodium gluconate and/or potassium gluconate may be reasonablycontemplated. Where the latter method is employed, supposing that thefood or food material contains glucose, an enzyme such as glucoseoxidase or a gluconic acid-producing microorganism may be used on thefood or food material to convert to gluconic acid. In the event the foodor food material contains sucrose or starch, a method that can be usedcomprises causing an enzyme such as invertase or amylase to produceglucose and, then, converting the glucose to gluconic acid in the samemanner as above.

The food and food materials in which gluconic acid, a nontoxic saltthereof and/or glucono-δ-lactone can be contained are not limited to theabove-mentioned kinds of foodstuffs but encompasses the entire spectrumof foods and food materials including sweeteners, honey, royal jelly,dairy products, soybean products, table salt, acidulants, condiments,starch, dextrin, processed animal or fish meat, noodles, beverages,bread, cakes, confectionaries, salted foods such as pickles, pH controlagents, freezing-point depressants for controlled freezing-pointstorage, moisture activity depressants, preservatives, excipients,diluents/volume builders, and so on. Some representative foods and foodmaterials, among those mentioned above, are now explained.

(1) Honey

Honey contains a small amount of gluconic acid but in order that thebifidobacterial growth promoting activity of gluconic acid may beovertly expressed, its concentration must be increased. In considerationof the fact that the sweet taste of honey blends well with the sourtaste of gluconic acid, a novel type of honey with a hint of sour tastecan be produced by adding gluconic acid or glucono-δ-lactone to honey orconverting the glucose occurring in honey enzymatically to gluconicacid.

(2) Sweeteners

Sweeteners such as isomerized syrup, starch syrup, brown sugar andglucose are frequently used in beverages, candies and confections. Thesesweeteners can be modified and converted to novel functional sweetenershaving a sour taste by adding gluconic acid and/or glucono-δ-lactone orconverting their ingredients partly to gluconic acid.

(3) Oligosaccharides

Oligosaccharides having various functional characteristics have beendeveloped. By adding gluconic acid, a nontoxic salt thereof and/orglucono-δ-lactone to such oligosaccharides to amplify theirfunctionalities, novel gustatory sweeteners can be provided.

(4) Dairy Products

Milk and skim milk are in use as raw materials for a variety of foods.By adding gluconic acid, they can be made into novel gustatoryfunctional food materials.

Aside from the above mentioned uses, gluconic acid, a nontoxic saltthereof and/or glucono-δ-lactone may be included in all kinds of foodsand food materials and can be used selectively according to objectives,e.g. addition of a sour or salty taste or for calcium or magnesiumsupplementation.

Moreover, alkali metal salts of gluconic acid which, as aforesaid, havebifidobacterial growth promoting activity possess for the followingadditional effects.

(1) When used in combination with aspartame, they improve the sweetnessof aspartame.

(2) When used in combination with a coagulant for the production of asoybean curd, they do not influence on the flavor and texture of thesoybean curd.

(3) When used in combination with common salt, they improve the taste ofsodium chloride and do not deteriorate its preservative effect.

Furthermore, gluconic acid has an action to impart a function toacidulants heretofore in use without deteriorating their nativegustatory quality, flavor and intensity of sour taste.

These aspects are now explained in detail.

A functional sweetener containing aspartame and an alkali metalgluconate:

Aspartame (α-L-aspartyl-L-phenylalanine methyl ester; hereinafterreferred to as aspartame) is about 100-200 times as sweet as sucrose andwhen used in food, its low energy feature can be exploited withadvantage and, hence, aspartame is in broad use as a diet sweetener in avariety of beverages and cakes, among others. Its quality of sweetnessis comparatively akin to that of sucrose but aspartame has the defectthat its sweetness is slightly delayed in onset and tends to remainlonger on the tongue. Therefore, means for ameliorating these aspectshave been explored. For example, the use of aspartame in combinationwith cyclodextrin (JP Kokai S-60-114166) and the use of aspartame incombination with corn syrup (JP Kokai S-60-114167) are known. However,since cyclodextrin and corn syrup are digested and absorbed, thusfunctioning as energy sources, these methods are not satisfactory fromthe consideration of low energy formulation.

Meanwhile, it is known that alkali metal salts of gluconic acid improvethe taste of the synthetic sweetener acesulfame K (JP Kokai S-59-66857)but the literature is reticent about the ways in which an alkali metalgluconate actually improves the taste.

The inventors explored for means by which the gustatory quality ofaspartame may be brought as close as possible to that of sucrose withoutdetracting from its characteristics and found that the above object canbe accomplished by using aspartame in combination with an alkali metalsalt of gluconic acid. Thus, an alkali metal gluconate added in a smallamount improves the quality of sweetness of aspartame so that there isprovided a sweetener composition comprising aspartame and alkali metalgluconate which has a low caloric value and yet encourages selectivegrowth of bifidobacteria, thus being very satisfactory from healthpoints of view and, hence, of value as a functional sweetener.

The functional sweetener of this invention can be used for sweeteningall types of foods.

Regarding the amount of such alkali metal gluconate, an improving effecton the quality of sweetness can be obtained at the level of addition ofat least one equivalent with respect to each weight part of aspartamebut when the added amount of the alkali metal gluconate is tooexcessive, a salty taste will be developed. Therefore, the level ofaddition should be adjusted according to each type of food. For example,in the case of certain foods in which salty taste is disagreeable, suchas saft drinks, the concentration of the alkali metal gluconate thereinis preferably not higher than 1%. Conversely in the case of foods whichshould be both sweet and salty taste, such as pickles, it is notobjectionable at all even if the concentration of the alkali metalgluconate exceeds 1%.

Regarding the mode of use of the sweetener of this invention, aspartameand an alkali metal gluconate may be independently added to food oralternatively a premix of aspartame and alkali metal gluconate, preparedfor the particular kind of food, may be added to the food. As to theapplication form of the sweetener, ordinary application forms such aspowders, granules, liquids etc. can be utilized. It is also permissibleto incorporate various auxiliary components such as acidulants, othersweeteners, condiments, excipients, etc. in addition to aspartame andalkali metal gluconate.

The functional sweetener of this invention is such that the taste ofaspartame has been brought as close as possible to that of sucrose andthat it is not only an excellent low-energy sweetener but also afunctional sweetener because it provides for selective growth ofintestinal bifidobacteria as the effect of the alkali metal gluconatecontained.

A functional coagulant prepared by adding an alkali metal gluconate tothe coagulant for soybean curds:

While a soybean curd is manufactured by adding a coagulant to soy milkfrom soybeans, the conventional coagulant, glucono-δ-lactone isdisadvantageous in that, when dissolved in water, it is hydrolyzed togluconic acid so that when this coagulant is used in excess, a sourtaste is imparted to the product curd. In this sense, the highestpermissible level of addition is considered to be 0.3%.

Development of functional foods is in progress to keep abreast of theincreasingly health-oriented mind of the public and, if only because itis a traditional food, it should be of great significance if soybeancurds could be provided with a new function.

Based on the above-mentioned new finding that gluconic acid or itsequivalent has activity to promote growth of bifidobacteria, theinventors of this invention did much research to accomplish theabovementioned object and found that by adding an alkali metal salt ofgluconic acid in the course of production of a soybean curd, afunctional soybean curd containing gluconic acid or its equivalent in ahigh concentration can be manufactured without detracting from thenative flavor and texture of soybean curds. As coagulant for soybeancurds that can be used, there can be mentioned the conventionalcoagulants for soybean curds, for example glucono-δ-lactone, calciumgluconate, calcium sulfate, and magnesium chloride, inclusive of theirpremixes.

The ratio of alkali metal gluconate to coagulant in the functionalcoagulant which can be prepared by adding the alkali metal gluconate tothe conventional coagulant for soybean curds can be generally about 0.3to 30 parts to each part of the coagulant.

In manufacturing a soybean curd using such a functional for coagulantsoybean curd containing an alkali metal gluconate, the amount of saidconventional coagulant can be just enough to cause coagulation of soymilk but it is generally preferable that the amount of gluconic acid inthe product soybean curd be not less than 0.5% (% by weight; the sameapplies hereinafter).

Furthermore, in the manufacture of a soybean curd, salts of otherorganic acids, such as sodium malate, sodium citrate, etc., aresometimes used in addition to the coagulant.

The level of addition of alkali metal gluconate is not so strict butwhen functionality is taken into consideration, it is preferably addedat a final concentration of at least 0.5% as gluconic acid in thesoybean curd. Where glucono-δ-lactone and/or calcium gluconate or acomposition containing either of them or both is used as the coagulant,the amount of alkali metal gluconate is preferably adjusted so that thetotal concentration as gluconic acid will be at least 0.5%.

The alkali metal gluconate can be added in any appropriate stage ofsoybean curd production. One examplary method of addition comprisesadding said salt to the "go" obtained by milling soybeans in water or tothe soy milk available on removal of insoluble residue following theheat-treatment of "go". As to the manner of addition, the gluconate maybe added either singly or in the form of a premix containing variousadditives such as the antifoam and coagulant, which are commonlyemployed in the manufacture of soybean curds, and/or other foodmaterials.

The alkali metal gluconate can be used in all types of soybean curds,including "kinugoshi tofu", "prepacked tofu", "momen tofu", "soft tofu"and so on. In each case, the coagulants (e.g. glucono-δ-lactone, calciumsulfate, magnesium chloride, etc.) and other additives which areconventionally used in the production of the respective types of soybeancurds can be used.

The functional soybean curd of this invention is of value in the sensethat by adding a gluconic acid compound such as an alkali metalgluconate to the conventional soybean curd, the soybean curd is providedwith a new function of promoting growth of bifidobacteria withoutaffecting the inherent flavor and texture of soybean curds.

A functional salt prepared by adding an alkali metal gluconate to commonsalt:

It is said that the recent increase in the incidence of various adultdiseases is significantly related to dietary life and, as one of theetiologic factors involved, a causal relationship of sodium chlorideintake to hypertension has been pointed out. As a countermeasure, avariety of low-salt foods have appeared on the market for reducingsodium chloride intake. However, it is not true that sodium chloride isused for the sole purpose of adding a salty taste to food but, in manyinstances, the salt plays significant roles in the processing andpreservation of food. For example, warnings are being voiced against theill effect of reduced salt contents on the processing characteristicsand shelf-life of pickles owing to the insufficient osmolarity andincreased water activity.

To overcome the above disadvantage, such means as using potassiumchloride to make up for a reduction in sodium chloride content or addingan acidulant, such as vinegar or an organic acid, have been practicedbut all affect the taste of food significantly and, therefore, none canbe said to be effective means. Under the circumstances, development ofan additive capable of reducing the sodium chloride without affectingthe taste and shelf-life of salt-formulated foods has been awaited inearnest.

As the result of the intensive research done for solving the aboveproblems, it was found that although alkali metal salts of gluconic acidare less salty than sodium chloride, their taste is quality-wise closeto that of sodium chloride so that these salts can be effectivesubstitutes for common salt. Moreover, it was discovered, as mentionedabove, that the gluconic acid compound is only sparingly absorbed in thesmall intestine and mostly reaches the large intestine where itselectively promotes growth of bifidobacteria, suggesting that it is notonly of use as a substitute for sodium chloride but is beneficial inthat it controls intestinal function. This invention provides a novelsalting agent having the function of promoting growth of bifidobacteriawithout detracting from the quality of saltiness and shelf-life ofsalt-formulated foods.

The amount of alkali metal gluconate is not so critical. Thus, even ifhalf or more of the usual amount of sodium chloride is replaced with analkali metal gluconate, there is no untoward effect on the taste of foodand at a substitution rate of 40% or more the gluconate rounds off thetaste of sodium chloride so that its addition is effective in thisrespect, too.

With regard to the manner of use, it may be added independently ofcommon salt or in the form of a premix. In addition, various othercomponents such as acidulants, condiments, sweeteners and excipients canalso be included in formulations.

The relative intensities of saltiness of sodium gluconate and potassiumgluconate are shown below.

Their amounts to be added can be determined by consulting these valuesaccording to the intended intensity of saltiness of the product food.

                  TABLE 1                                                         ______________________________________                                        Intensity of salty taste                                                      (calculated from the threshold value)                                                       Saltiness index*                                                ______________________________________                                        Sodium chloride 1                                                             Sodium gluconate                                                                              0.2                                                           Potassium gluconate                                                                           0.15                                                          ______________________________________                                         *The relative value with the intensity of saltiness of sodium chloride        being taken as unity.                                                    

In using an alkali metal gluconate as a salting (saltiness-imparting)agent, its specific uses are virtually unlimited and it can be used inall kinds of foods in which sodium chloride is conventionallyformulated. Thus, for example, pickles, noodles, bread, "miso", soysource, salted fish or shellfish, soups, "miso" soup, etc. can bementioned. Moreover, if the sole object is to impart saltiness to food,it is not always necessary to use sodium chloride in conjunction but analkali metal gluconate alone can be added.

The functional salt of this invention is of value as a functionalsaltiness in the sense that an alkali metal gluconate added to food invarious uses for which sodium chloride is conventionally added providesfor an additional function of promoting growth of bifidobacteria withoutdetracting from the quality of saltiness and shelf-life of the food.

A functional acidulant prepared by adding gluconic acid to an acidulant:

As substances which impart sourness to food, that is to say acidulants,a variety of organic acids have been employed but since each of them hasits own taste and intensity of sourness, these acidulants are beingselectively used according to types of foods. Thus, they are used inways taking advantage of their respective characteristics; for examplecitric acid is frequently used in juices, tartaric acid in candies, andacetic acid in pickles. Moreover, in some types of foods, more than oneacidulants are used in combination.

While development of functional foods is the order of the day, betteracidulants would be provided if new functions could be added to theconventional acidulants as well.

The inventors of this invention did much research to that end and foundthat by adding gluconic acid to the conventional acidulant on thestrength of the above-mentioned discovery of the growth promoting effectof gluconic acid on bifidobacteria, the new function can be imparted tothe acidulant without adversely affecting the inherent characteristics,namely taste, flavor and sourness, of said acidulant. This finding wasfollowed by further investigations which have resulted in thedevelopment of this invention herein disclosed.

This invention provides a novel acidulant having the function ofpromoting growth of bifidobacteria without detracting from the taste,flavor and intensity of sourness of the conventional acidulant throughpartial substitution of gluconic acid for said acidulant and a foodcontaining said novel acidulant.

Gluconic acid for use in this invention includes, besides gluconic acidas such, any substance that gives gluconic acid in aqueous solution, forexample glucono-δ-lactone, glucono-γ-lactone and alkyl esters ofgluconic acid, such as ethyl gluconate. Generally, however, gluconicacid and glucono-δ-lactone, both of which are approved food additives,are preferably employed.

As to the acidulant, this invention is applicable to all types ofacidulants heretofore in use as food acidulants, namely citric acid,lactic acid, tartaric acid, malic acid, acetic acid and succinic acid,to name but a few.

For partial substitution of gluconic acid for such an acidulant, theirratio depends on the type and intensity of sourness (degree of sourness)of the acidulant. The upper limit substitutability values for therespective organic acids (the relative amounts of gluconic acid that canbe added without affecting the taste, flavor and intensity of sournessof the conventional acidulants) are shown on an equivalent sournessbasis below (Table 2). The values shown correspond to cases in whichgluconic acid as such is added and when any of said gluconic acidprecursors is employed, the amount of gluconic acid formed in aqueoussolution should be used as the basis of conversion. For reference'ssake, the degrees of sourness of various acidulants with the degree ofsourness of gluconic acid being taken as unity are shown (Table 3).

                  TABLE 2                                                         ______________________________________                                                    Upper limit substitutability                                                  values of gluconic acid                                           Acidulant   (on an equivalent sourness basis)                                 ______________________________________                                        Citric acid 40%                                                               Lactic acid 30%                                                               Tartaric acid                                                                             40%                                                               Malic acid  30%                                                               Acetic acid 20%                                                               Succinic acid                                                                             50%                                                               ______________________________________                                    

                  TABLE 3                                                         ______________________________________                                        Organic acid  Degree of sourness                                              ______________________________________                                        Gluconic acid 1                                                               Citric acid   3                                                               Lactic acid   3.2                                                             Tartaric acid 4.7                                                             Malic acid    4.1                                                             Acetic acid   4                                                               Succinic acid 3.6                                                             ______________________________________                                    

The manner of reading the above tables is now explained taking citricacid as an example.

It can be seen from the above tables that when citric acid is added inan amount corresponding to 60% of its specification amount and gluconicacid is added in an amount corresponding in sourness to 40% of saidspecification amount, the result is not much different from the resultobtainable by singular use of citric acid in terms of taste, flavor, andintensity of sourness. For example, for 40% replacement of the sournessof 0.5% citric acid with gluconic acid, citric acid and gluconic acidcan be added at concentrations of 0.3% and 0.6%, respectively.

With regard to the manner of use of gluconic acid, it can be addedindependently of various acidulants or used in the form of a premix. Thepercentage of gluconic acid in such usage is preferably not greater thanthe value shown in terms of the degree of sourness in the above table.If gluconic acid is used in an amount exceeding the above value, it mayhappen that the taste and flavor of the acidulant are modified so thatits native characteristics may not be exploited. Moreover, when morethan one acidulants are used in combination, the formulation amount canbe calculated with reference to the above-mentioned upper limit gluconicacid-substitutability values based on the degrees of sourness of therespective acidulants.

Then, as the most desirable examples of the functional acidulantobtainable by adding gluconic acid to a conventional acidulant, theacidulant comprising citric acid and gluconic acid in a ratio of10:3˜10:20, the acidulant comprising tartaric acid and gluconic acid ina ratio of 10:5˜10:30, and the acidulant comprising lactic acid andgluconic acid in a ratio of 10:3˜10:15 can be mentioned. Theseacidulants can be used singly or as a combination of two or more speciesaccording to the substrate food. For example, when one or more of theseacidulants are to be added to a juice or a candy, the level of additionmay be not less than 0.5% by weight.

The functional acidulant of this invention comprising a conventionalacidulant and gluconic acid is of value as a function-oriented acidulanthaving an additional function of promoting growth of bifidobacteriawithout detracting from the well-known characteristic required ofacidulants, whether in taste, in flavor or in the intensity of sourness.

Some test examples and working examples of this invention are presentedbelow.

TEST EXAMPLE 1 Gluconic Acid Absorption Test by the Intestinal LoopMethod

The test was performed using sodium gluconate as an example of thisinvention and glucose as a control. As experimental animals, a total of12 rats (Wistar strain, male, aged 7 weeks, body weights 280 g, approx.)were divided into 4 groups.

Each experimental rat was fasted for 24 hours and then, under etheranesthesia, a loop about 10 cm long was formed of the upper part of thesmall intestine (the lower part of jejunum below the ligament of Treitz)or the lower part of the small intestine (ileum). The loop was infusedwith 0.5 ml of 100 mM sodium gluconate-saline or glucose-saline and theintestine was closed for 30 minutes' absorption. The loop was thenexcised and the internal fluid was washed out with 20 ml of saline.

The residual gluconic acid in the saline wash was assayed using F-KitD-Gluconic Acid (Boehringer-Mannheim-Yamanouchi) and the glucose wasassayed with Glucose C II Test Wako (Wako Pure Chemicals) and therespective values were recorded as dose-recovery amounts.

Moreover, on the occasion of said excision of the loop, the adjacentportion of the small intestine was additionally excised to prepare aloop (about 10 cm) for an addition-recovery experiment. This loop wasinfused with 0.5 ml of the test solution and the internal liquid wasrecovered and determined for gluconic acid or glucose in the same manneras above. Using the value thus found as the addition-recovery amount,the percent residue of each sample was calculated by means of thefollowing equation.

    ______________________________________                                        Residue (%) =                                                                              [(dose-recovery amount)/                                                      (addition-recovery amount)] × 100                          ______________________________________                                    

                  TABLE 4                                                         ______________________________________                                        Part of small intestine                                                                        Upper part                                                                              Lower part                                         ______________________________________                                                       Glucose                                                        Residue (%)*     0 ± 0  50.7 ± 6.0                                                     Sodium gluconate                                               Residue (%)*     80.1 ± 4.8                                                                           89.4 ± 13.4                                     ______________________________________                                         *Mean ± S.D.                                                          

The percent residues in the upper and lower parts of the small intestineare shown in Table 4. It is apparent from Table 4 that about 80-90% ofgluconic acid remained in the small intestine, suggesting that thiscompound was only partly digested and absorbed in the small intestine.In contrast, glucose was almost completely absorbed in the upper part ofthe small intestine and little reached the large intestine so that itcannot be expected to encourage growth of bifidobacteria.

TEST EXAMPLE 2 Selective Utilization of Gluconate

As shown in Table 5, Bifidobacterium adolescentis ATCC 15703 andBifidobacterium adolescentis ATCC 15705 were used as representativebifidobacteria, Welch's bacillus (Clostridium perfringens GKK 16 andClostridium perfringens CWiu) as harmful bacteria, and the genusBacteroides (Bacteroides fragilis W-7) as dominant intestinal bacteria.GAM broth (Nissui Pharm. Co., Ltd.) was inoculated with the test strainand incubated anaerobically at 37° C. for 20 hours to prepare apreculture.

As test substances, sodium gluconate, fructooligo-saccharide and glucosewere used (Table 5). For use as the glycolysis test medium, the basalmedium available on elimination of agar from the 1/2 formulation of theGAM semisolid without dextrose medium (Nissui Pharm. Co., Ltd.) wassupplemented with 0.5 w/v % of the test substance and adjusted to pH6.9.

The above test medium was inoculated with 0.01 v/v % of said precultureand incubated anaerobically at 37° C. for 20 hours. Then, the opticaldensity (660 nm) and pH were determined. The medium was distributed in 5ml aliquots into test tubes sized 18 mm in diameter. Anaerobic culturewas carried out using Anaeropak (Mitsubishi Gas Chemical Co.).Measurement of optical density (OD) was performed over the test tubeusing Shimadzu Milton-Roy spectrophotometer "Spectronic 20A". The pH wasdirectly measured using the glass electrode of the pH Meter HM-30S (ToaDempa Kogyo). The result was compared with that of the basal mediumcontrol and the judgement that the test substance was utilized was madein the case of an increase in optical density (OD) or a depression ofpH. The results are shown in Table 5.

In addition, using Bifidobacterium adolescentis ATCC 15703 as arepresentative strain, a similar test was performed for calciumgluconate. The results are shown in Table 6.

                  TABLE 5                                                         ______________________________________                                                           Test substance                                                                      Control                                                                       (basal   Sodium                                      Test strain              medium)  gluconate                                   ______________________________________                                        Bifidobacterium OD       0.27     0.68                                        adolescentis ATCC 15703                                                                       pH       6.04     5.22                                        Bifidobacterium OD       0.15     0.66                                        adolescentis ATCC 15705                                                                       pH       6.10     5.28                                        Clostridium perfringens                                                                       OD       0.27      0.23*                                      GKK 16          pH       6.46     6.43                                        Clostridium perfringens                                                                       OD       0.36      0.33*                                      CWiu            pH       6.60     6.56                                        Bacteroides fragilis                                                                          OD       0.48     0.48                                        W-7             pH       6.12     6.07                                        ______________________________________                                                           Test substance                                                                      Fruct-                                                                        oligosac-                                            Test strain              charide  Glucose                                     ______________________________________                                        Bifidobacterium OD       0.77     0.84                                        adolescentis ATCC 15703                                                                       pH       4.32     4.23                                        Bifidobacterium OD       0.80     0.76                                        adolescentis ATCC 15705                                                                       pH       4.27     4.27                                        Clostridium perfringens                                                                       OD       0.40     0.95                                        GKK 16          pH       6.01     4.99                                        Clostridium perfringens                                                                       OD       0.47     1.04                                        CWiu            pH       6.53     5.24                                        Bacteroides fragilis                                                                          OD       0.64     0.60                                        W-7             pH       5.20     4.99                                        ______________________________________                                         *Decrease in OD as compared with control                                 

                  TABLE 6                                                         ______________________________________                                                           Test substance                                                                  Control                                                                       (basal   Calcium                                         Test strain          medium)  gluconate                                       ______________________________________                                        Bifidobacterium OD       0.25     0.45                                        adolescentis ATCC 15703                                                                       pH       6.81     5.39                                        ______________________________________                                    

It is apparent from Tables 5 and 6 that sodium gluconate and calciumgluconate both according to this invention are well utilized bybifidobacteria and that sodium gluconate is not assimilated but ratherinhibits growth of Welch's bacillus. It is also clear that sodiumgluconate is not utilized by bacteria of the genus Bacteroides which aredominant members of the intestinal flora, indicating that the growthfactor of this invention is an excellent selective growth promotant. Onthe other hand, glucose is well utilized by bifidobacteria but, unlikethis invention, utilized without selectivity. Moreover, glucose isalmost completely absorbed in the upper part of the small intestine andcannot reach the large intestine as can be seen from Test Example 1,thus being not considered to be a satisfactory bifidus factor.

TEST EXAMPLE 3 Effect of Glucono-δ-lactone on Human Fecal Flora

Glucono-δ-lactone (a lactone of gluconic acid), an example of thisinvention, was administered to man and its effect on the intestinalflora and stool properties was investigated.

One healthy male adult volunteer (50 years old) was asked to ingest 9 gof glucono-δ-lactone daily in 3 divided doses and the intestinal florain the stool was determined on day 0, day 10 and day 30 of ingestion andday 20 of washout after ingestion in accordance with the method ofMitsuoka (Tomotari Mitsuoka: Chonaikinno-Sekai (A Color Atlas ofAnaerobic Bacteria), Sobunsha, Tokyo, p. 53, 1984)). The results are setforth in Table 7. As to stool properties, the subject himself made asensory evaluation of consistency, color and odor on a daily basis.

                  TABLE 7                                                         ______________________________________                                                  Logarithm of the viable count                                                 in each gram of feces                                                         The figure in parentheses represents                                          the percentage (%) of the total count                                           Day 0    Day 10   Day 30 Day 20 of                                Microorganism                                                                             of intake                                                                              of intake                                                                              of intake                                                                            washout                                  ______________________________________                                        Bifidobacterium                                                                           9.5      0.5      0.7    9.7                                                  (10)     (65)     (94)   (19)                                     Bacteroidaceae                                                                            10.2     10.0     9.3    10.3                                                 (52)     (22)      (4)   (69)                                     Eubacterium 9.4      9.6      8.9    9.5                                                   (8)      (8)      (1)   (11)                                     Peptococcaceae                                                                            9.9      9.3      8.7    8.7                                      (anaerobic strepto-                                                                       (30)      (5)      (1)    (2)                                     cocci)                                                                        C. perfringens                                                                            7.0      <3       <3     3.6                                      (Welch's bacillus)                                                            Enterobacteriaceae                                                                        7.0      6.4      6.2    6.8                                      (inclusive of                                                                 coliform bacteria)                                                            Total viable count                                                                        10.5     10.6     10.7   10.4                                                 (100)    (100)    (100)  (100)                                    ______________________________________                                    

It is apparent from Table 7 that the population of bifidobacteriainhabiting the intestinal tract was remarkably increased byglucono-δ-lactone. In contrast, the viable count of bacteria of thefamily Bacteroidaceae was decreased and the count of Welch's bacillusalso fell below the detection limit. The stool was softened, intestinemovement improved, and fecal odor suppressed.

TEST EXAMPLE 4 Attenuation of Canine Fecal Odor

Glucono-δ-lactone, as an example of this invention, was administered todogs and its fecal deodorizing effect was evaluated.

Eight male beagle dogs aged about 10 months (body weights 10-13 kg) weredivided into two groups of 4 to provide a glucono-δ-lactone treatmentgroup and a glucono-δ-lactone-free control group.

Glucono-δ-lactone was filled in gelatin capsules and administered in adose of 50 mg/kg body weight 3 times a day at 10 o'clock, 13 o'clock and16 o'clock for 2 consecutive weeks. Before administration and at 2 weeksof administration, fresh feces in the treatment group and control groupwere respectively collected and the fecal odor was organolepticallyevaluated by the sensory test.

Attenuation of fecal odor was obtained in the glucono-δ-lactonetreatment group.

TEST EXAMPLE 5 Sensory Test of Sweeteners

The degree of improvement in sweetness on addition of sodium gluconateto aspartame was evaluated by a sensory test.

Method of the sensory test

A 10% solution of sucrose (x), a 0.083% solution of aspartame (y), and asolution (z) prepared by adding 0.3% of sodium gluconate to a 0.083%solution of aspartame were paired and it was evaluated how much thetaste (A) of the first-tasted solution was close to the taste of sucroseas compared with the taste (B) of the second-tasted solution inaccordance with the following criteria. It was ensured that theintensity of sweetness would be uniform over x, y and z and a 10%solution of sucrose was used as control.

The panel consisted of 10 tasters

    ______________________________________                                        Compared with B, A is                                                         very close to control                                                                            +3                                                         fairly close to control                                                                          +2                                                         slightly close to control                                                                        +1                                                         Eqivocal            0                                                         Compared with A, B is                                                         slightly close to control                                                                        -1                                                         fairly close to control                                                                          -2                                                         very close to control                                                                            -3                                                         ______________________________________                                    

Result

                  TABLE 8                                                         ______________________________________                                        First   Second  +3       +2   +1      0   -1                                  ______________________________________                                        x       y       5        3    2       0   0                                   y       x       0        0    0       0   1                                   x       z       0        2    7       0   1                                   z       x       0        0    2       0   6                                   y       z       0        0    1       0   1                                   z       y       4        3    1       1   1                                   Total       9        8      13      1   10                                    ______________________________________                                                                       Total Average                                  First  Second    -2     -3     score score                                    ______________________________________                                        x      y         0      0      23    2.3                                      y      x         4      5      -24   -2.4                                     x      z         0      0      10    1.0                                      z      x         2      0      -8    -0.8                                     y      z         6      2      -18   -1.8                                     z      y         0      0      18    1.8                                      Total        12     7                                                         ______________________________________                                    

Analysis of variance of the above data showed significant differencesamong x, y and z but the quality of sweetness of (z), i.e. aspartameplus sodium gluconate, was significantly close to that of sucrose (x) ascompaired with aspartame alone (y), indicating an overt taste-improvingeffect. The relationship of the tastes of sucrose, aspartame andaspartame plus sodium gluconate is diagrammatically illustrated below.

    ______________________________________                                                              Aspartame +                                                                   sodium                                                  Aspartame             gluconate  Sucrose                                      (y)                   (z)        (x)                                          ↓ -1.38        ↓ 0.30                                                                            ↓ 1.08                                ______________________________________                                        -1.5   -1.0       -0.5   0      0.5  1.0                                      ______________________________________                                    

For example, when the relationship of these three tastes is expressed byassigning 100 to the taste of sucrose and 0 to the taste of aspartame,the taste of aspartame plus sodium gluconate is 68, which means a markedimprovement.

EXAMPLE 1 (sweetener)

An orange fruit drink was prepared by adding water, citric acid and asweetener composed of aspartame and alkali metal gluconate to acommercial 100% orange juice. As a comparison example, an orange juicedrink was similarly prepared using a sufficient amount of granulatedsugar to provide the same degree of sweetness. Then, the difference intaste between them was evaluated by the sensory test (triangle test). Asa result, no significant difference was found between the invention andthe comparison example.

    ______________________________________                                                   Formulation Formulation                                                       of Example  of Comparison                                                    (A)      (B)     Example                                            ______________________________________                                        100% Orange juice                                                                         150 g      150 g   150 g                                          Citric acid  3.6 g      3.6 g   3.6 g                                         Aspartame   0.22 g     0.22 g  --                                             Sodium gluconate                                                                           2.1 g     --      --                                             Potassium gluconate                                                                       --          5.0 g  --                                             Granulated sugar                                                                          --         --       51 g                                          Water       516 g      516 g   516 g                                          ______________________________________                                    

Sensory test

The panel consisted of 10 panelists.

                  TABLE 9                                                         ______________________________________                                        Test group           Sensory test.sup.Note)                                   ______________________________________                                        Example A                                                                              ←→                                                                        Comparison  5/10  (no significant                                             Example           difference)                                Example B                                                                              ←→                                                                        Comparison  5/10  (no significant                                             Example           difference)                                ______________________________________                                         .sup.Note) Number of panelists giving the correct answer                      (discrimination)/number of panelists                                     

EXAMPLE 2 (Honey)

To 200 g of Chinese milk vetch honey was added 300 g of water followedby addition of 0.5 g of an enzyme preparation containing glucose oxidase(60 units/mg) and catalase (390 units/mg) activities and the reactionwas carried out at 23° C. under aerobic conditions for 90 minutes toprovide a gluconic acid-containing honey. The result of assay ofgluconic acid in this reaction mixture by high performance liquidchromatography is shown in Table 10. This product has a refreshinggustatory quality possessing both a sour taste and a sweet taste.

                  TABLE 10                                                        ______________________________________                                                         Before After                                                                  reaction                                                                             reaction                                              ______________________________________                                        Gluconic acid content (%)                                                                        0.05     3.5                                               pH                 4.2      2.6                                               ______________________________________                                    

EXAMPLE 3 (Isomerized syrup)

To 135 g of isomerized syrup (42% fructose grade) was added 300 g ofwater followed by addition of 0.5 g of the same enzyme preparation asused in Example 2. The reaction was conducted aerobically at 23° C. for90 minutes to provide a gluconic acid-containing isomerized syrup. Theresult of assay of gluconic acid in this isomerized syrup by highperformance liquid chromatography is shown in Table 11.

                  TABLE 11                                                        ______________________________________                                                         Before After                                                                  reaction                                                                             reaction                                              ______________________________________                                        Gluconic acid content (%)                                                                        0.02     3.5                                               pH                 5.8      2.5                                               ______________________________________                                    

For the production of a gluconic acid-containing isomerized syrup, it isan alternative reasonable procedure to conduct the above reaction uponcompletion of conversion of glucose to fructose in the process ofisomerized syrup production and, thereafter, concentrate the reactionproduct.

EXAMPLE 4 (Glucose)

In 400 g of water was dissolved 100 g of glucose followed by addition of0.5 g of the same enzyme preparation as used in Example 2. The reactionwas then conducted aerobically at 23° C. for 90 minutes to provide agluconic acid-containing glucose syrup. The result of assay of gluconicacid in this glucose syrup by high performance liquid chromatography isshown in Table 12.

                  TABLE 12                                                        ______________________________________                                                         Before After                                                                  reaction                                                                             reaction                                              ______________________________________                                        Gluconic acid content (%)                                                                        0        5.4                                               pH                 6.7      2.6                                               ______________________________________                                    

EXAMPLE 5 (Corn syrup )

To 100 g of corn syrup was added 400 g of water followed by addition ofthe same enzyme preparation as used in Example 2 and the reaction wascarried out aerobically at 23° C. for 90 minutes to provide a gluconicacid-containing corn syrup. The result of assay of gluconic acid in thiscorn syrup by high performance liquid chromatography is shown below inthe table. As in Example 3, it is also a reasonable procedure to conductthe above reaction upon completion of hydrolysis of starch in theprocess of corn syrup production to theraby provide a gluconicacid-containing corn syrup.

                  TABLE 13                                                        ______________________________________                                                         Before After                                                                  reaction                                                                             reaction                                              ______________________________________                                        Gluconic acid content (%)                                                                        0.05     3.0                                               pH                 4.2      2.8                                               ______________________________________                                    

EXAMPLE 6 (Oligosaccharide)

To 150 g of isomaltooligosaccharide (Isomalt 500, Showa Sangyo) wasadded 350 g of water followed by addition of 0.5 g of the same enzymepreparation as used in Example 2 and the reaction was conductedaerobically at 23° C. for 90 minutes to provide a gluconicacid-containing oligosaccharide syrup. The result of assay of gluconicacid in this oligosaccharide syrup by high performance liquidchromatography is shown in Table 14. As mentioned in Example 3, it isalso a reasonable procedure to first conduct theisomaltooligosaccharide-producing enzymatic reaction in the process ofoligosaccharide production, then conduct the above-described reaction,and finally concentrate the reaction mixture. Aside from the foregoing,fructooligosaccharide, galactooligosaccharide, lactosucrose, etc. canalso be converted to the corresponding gluconic acid-containingoligosaccharides.

                  TABLE 14                                                        ______________________________________                                                         Before After                                                                  reaction                                                                             reaction                                              ______________________________________                                        Gluconic acid content (%)                                                                        0.02     4.0                                               pH                 3.6      2.5                                               ______________________________________                                    

EXAMPLE 7 (Brown sugar)

To 100 g of brown sugar was added 400 g of water. Then, 0.1 g ofinvertase (4 U/ml) and 0.5 g of the same enzyme preparation as used inExample 2 were added and the reaction was conducted aerobically at 23°C. for 90 minutes to provide a gluconic acid-containing brown sugarsyrup. The result of assay of gluconic acid in this brown sugar syrup byhigh performance liquid chromatography is shown in Table 15.

                  TABLE 15                                                        ______________________________________                                                         Before After                                                                  reaction                                                                             reaction                                              ______________________________________                                        Gluconic acid content (%)                                                                        0        3.6                                               pH                 5.8      3.0                                               ______________________________________                                    

EXAMPLE 8 (Milk beverage)

To 20 g of skim milk was added 180 g of water followed by addition of 2g of calcium gluconate and 3 g of sodium gluconate. This procedureprovided a stable calcium-enriched milk drink giving no precipitation onheating.

EXAMPLE 9 (Coagulated milk)

To 100 g of cow's milk were added 10 g of sugar and 3 g ofglucono-δ-lactone and the mixture was heated at 80° C. for 30 minutes tocoagulate the milk and provide a coagulated milk dessert food.

EXAMPLE 10 (Apple juice drink)

An apple juice was prepared by adding water, citric acid, granulatedsugar and sodium gluconate to a commercial 100% apple juice. (Theformulation is shown below).

Compared with the sodium gluconate-free juice, this product had a thicktaste close to the taste of 100% apple juice.

    ______________________________________                                        100% Apple juice       20 g                                                   Granulated sugar       8 g                                                    Citric acid            0.32 g                                                 Sodium gluconate       1.08 g                                                 Water                  80 g                                                   ______________________________________                                    

EXAMPLE 11 (Soybean curd)

To 200 g of soybeans which had been caused to absorb enough water byovernight soaking was added 350 ml of water and the mixture was milledin a mixer to prepare a "go". This "go" was steam-heated and after itstemperature had reached 100° C., was further heated for 3 minutes. The"go" was then immediately passed through a filter cloth to remove theinsoluble residues to provide a soy milk. To this soy milk was addedsodium gluconate at a level of 0.6% or 1.0% followed by addition of 0.3%of glucono-δ-lactone. The mixture was heated on a water bath at 80° C.for 30 minutes to provide a soybean curd.

The results are set forth in Table 16.

The sodium gluconate-containing soybean curd thus obtained was more"substantial" and had a better taste than a sodium gluconate-freecontrol soybean curd. Incidentally no remarkable difference was found inhardness and texture.

                                      TABLE 16                                    __________________________________________________________________________    Amount of sodium                                                                        Hardness.sup.Note 1)                                                                             Gluconic.sup.Note 2)                             gluconate (%)                                                                           (g/0.5 cm.sup.2)                                                                       Taste     acid content (%)                                 __________________________________________________________________________    Control   49       --        0.33                                             (not added)                                                                   0.6       50       More substantial                                                                        0.87                                                                than control                                               1.0       47       More substantial                                                                        1.23                                                                than control                                               __________________________________________________________________________     .sup.Note 1) Determined with a curd meter.                                    .sup.Note 2) As gluconic acid (calcd.)                                   

EXAMPLE 12 (Soybean curd)

A soy milk prepared in the same manner as in Example 11 was cooled to20° C. and sodium gluconate was added at a final concentration of 1%.Then, glucono-δ-lactone was added at a concentration of 0.3% and themixture was heated on a water bath at 90° C. for 40 minutes to provide asoybean curd.

The results are shown in Table 17.

The sodium gluconate-containing soybean curd thus obtained was more"substantial" and tasted better than the control soybean curd.Incidentally no remarkable difference was found in hardness and texture.

                  TABLE 17                                                        ______________________________________                                        Amount of                                                                     sodium     Hardness               Gluconic acid                               gluconate (%)                                                                            (g/0.5 cm.sup.2)                                                                        Taste        content (%)                                 ______________________________________                                        Control    57        --           0.33                                        (not added)                                                                   1.0        56        More substantial                                                                           1.23                                                             than control                                             ______________________________________                                    

EXAMPLE 13 (Soybean curd)

To a soy milk prepared in the same manner as in Example 11 was addedsodium gluconate at a concentration of 1.0%. Then, a coagulant composedof 67% glucono-δ-lactone and 33% calcium sulfate was added at the levelof 0.30 or 0.34%. The mixture was heated on a water bath at 80° C. for30 minutes to provide a soybean curd. As control, 0.30% of the samecoagulant as above was added to the sodium gluconate-free soy milk toprovide a control soybean curd.

The results are shown in Table 18.

The sodium gluconate-containing soybean curd was slightly softer but itshardness could be made close to that of control by increasing the amountof coagulant.

                                      TABLE 18                                    __________________________________________________________________________    Amount of sodium                                                                       Amount of            Gluconic acid                                   gluconate                                                                              coagulant                                                                            Hardness      content                                         (%)      (%)    (g/cm.sup.2)                                                                       Taste    (%)                                             __________________________________________________________________________    Control  0.30   40   --       0.22                                            (not added)                                                                   1.0      0.30   33   More substantial                                                                       1.12                                                                 than control                                             1.0      0.34   39   More substantial                                                                       1.15                                                                 than control                                             __________________________________________________________________________

EXAMPLE 14 (Coagulant)

Three (3) kg of sodium gluconate was mixed with 1 kg ofglucono-δ-lactone to provide 4 kg of a functional soybean curdcoagulant.

EXAMPLE 15 (Coagulant)

One (1) kg of potassium gluconate was mixed with 300 g ofglucono-δ-lactone to provide 1.3 kg of a functional soybean curdcoagulant.

EXAMPLE 16 (Coagulant)

three (3) kg of sodium gluconate was mixed with 600 g ofglucono-δ-lactone and 400 g of calcium sulfate to provide 4 kg of afunctional soybean curd coagulant.

EXAMPLE 17 (Soybean curd)

To a soy milk prepared in the same manner as in Example 11 was added thefunctional soybean curd coagulant obtained in Example 15 at aconcentration of 1.3% and the mixture was heated on a water bath at 80°C. for 30 minutes to provide a soybean curd. The soybean curd thusobtained had a very satisfactory taste without any deterioration inhardness, texture and other characteristics.

TEST EXAMPLE 6 (Sensory test of salting agents)

Common salt and a salting agent prepared by partial substitution ofsodium gluconate for common salt were used. The relative taste ofaqueous solutions of the respective materials as prepared to the sameintensity of saltiness was evaluated by a sensory test.

Method of the Sensory Test

An aqueous solution was prepared by dissolving the salting agentcontaining sodium gluconate (GNA) partially replacing common salt at afinal concentration of 5% and an aqueous solution of common salt wasalso prepared to the same intensity of saltiness. The difference intaste between these two aqueous solutions was evaluated by a triangletest.

The results are set forth in the following table.

                  TABLE 19                                                        ______________________________________                                                            Numer of Panelists                                                            giving the correct                                                            answer/total number of                                    Test group          panelists.sup.Note 1)                                     ______________________________________                                        Common  3.5%             Common                                               salt                ←→                                                                     salt   4/10                                          GNA    1.5%              3.8%                                                 Common  3.0%             Common                                               salt                ←→                                                                     salt   7/10*                                         GNA    2.0%              3.4%                                                 Common  2.5%             Common                                               salt                ←→                                                                     salt   8/10**                                        GNA    2.5%              3.0%                                                 Common  2.0%             Common                                               salt                ←→                                                                     salt   8/10**                                        GNA    3.0%              2.6%                                                 ______________________________________                                                            Quality of                                                Test group          saltiness.sup.Note 2)                                     ______________________________________                                        Common  3.5%             Common                                               salt                ←→                                                                     salt   --                                            GNA    1.5%              3.8%                                                 Common  3.0%             Common  GNA plus is milder 5                         salt                ←→                                                                     salt                                                 GNA    2.0%              3.4%   Common salt is milder 2                       Common  2.5%             Common  GNA plus is milder 7                         salt                ←→                                                                     salt                                                 GNA    2.5%              3.0%   Common salt is milder 1                       Common  2.0%             Common  GNA plus is milder 8                         salt                ←→                                                                     salt                                                 GNA    3.0%              2.6%   Common salt is milder 0                       ______________________________________                                         .sup.Note 1) *significant difference at 5% level                              **significant difference at 1% level                                          No mark: No significant difference                                            .sup.Note 2) Among the panelists giving the correct answer, the number of     penelists who answered that the GNA plus was milder or that common salt       was milder.                                                              

EXAMPLE 18 (Salting agent)

To 300 g of cucumbers were evenly smeared with 15 g of a salting agentcomposed of 40% sodium chloride and 60% alkali metal gluconate. Thecucumbers were then put in a tray and after placement of a cover inposition, a weight of 600 g was placed on the cover. The cucumbers wereallowed to stand in this condition at room temperature for 24 hours toprovide overnight-pickled cucumbers. As a comparison example,overnight-pickled cucumbers were similarly prepared using 15 g of sodiumchloride only as the salting agent. The overnight-pickled cucumbersobtained by using the gluconic acid salt was milder in saltiness thanthe comparison pickled cucumbers but there was no difference inappearance, texture, or degree of dehydration, indicating that thealkali metal gluconate can be a satisfactory substitute for common salt.

The results are set forth in Table 20.

                  TABLE 20                                                        ______________________________________                                        Raw material Overnight pickles                                                Salting agent                                                                              Cucumbers Yield    Taste etc.                                    ______________________________________                                        Common salt                                                                            6     g     300  g    260  g   Saltiness milder                      Sodium   9     g                        than Comparison                       gluconate                               Example.                                                                      No difference in                                                              texture.                              Common salt                                                                            6     g     300  g    263  g   Saltiness milder                      Potassium                                                                              9     g                        than Comparison                       gluconate                               Example.                                                                      No difference in                                                              texture.                              Common salt                                                                            15    g     300  g    264  g   --                                    (Comparison                                                                   Example)                                                                      ______________________________________                                    

TEXT EXAMPLE 7 (Acidulant)

As acidulants, citric acid, lactic acid, tartaric acid, malic acid,acetic acid and succinic acid were selected and each of these basalacidulants and an acidulant prepared by substituting gluconic acid forpart or all of the basal acidulant on an equivalent sourness basis wereused. The maximum amount of gluconic acid that could be formulated withsensory test was determined on the basis of sourness.

Method of the Sensory Test

Each acidulant as such (A) and an acidulant (B) prepared by substitutinggluconic acid for part or all of acidulant (A) on an equivalent sournessbasis were evaluated by the triangle test to see whether there was asensory test difference. The number of panelists was 10.

Results

The results are set forth in the following table.

                  TABLE 21                                                        ______________________________________                                               B                                                                             Degree of gluconic acid substitution                                          (% on a sourness basis)                                                A        20     30      40    50    60    100                                 ______________________________________                                        Citric acid                                                                            --     --      4/10  7/10* --    8/10**                              Lactic acid                                                                            --     4/10    7/10* --    --    8/10**                              Tartaric acid                                                                          --     --      1/10  7/10* --    8/10**                              Malic acid                                                                             --     4/10    7/10* --    --    8/10**                              Acetic acid                                                                            3/10   7/10*   --    --    --    10/10**                             Succinic acid                                                                          --     --      --    4/10  8/10**                                                                              10/10**                             ______________________________________                                         .sup.Note 1) Each FIG. in the table denotes the number of panelists givin     the correct answer (discrimination)/the total number of panelists.            *Significant difference at 5% level                                           **Significant difference at 1% level                                          No mark: No significant difference                                       

EXAMPLE 19 (Acidulant)

An orange juice was prepared by adding water, granulated sugar and, asacidulants, citric acid and gluconic acid to a commercial 100% orangejuice. As a comparison example, an orange juice drink was similarlyprepared by using citric acid as acidulant to provide for the equivalentdegree of sourness. The relative taste of both drinks was evaluated bythe sensory test (triangle test). No significant difference in taste wasfound between the two drinks.

    ______________________________________                                        Formulation of Example                                                        100% orange juice      150    g                                               Granulated sugar       51     g                                               Citric acid            2.16   g                                               50% Gluconic acid      8.64   g                                               Water                  509    g                                               Formulation of Comparison Example                                             100% orange juice      150    g                                               Granulated sugar       51     g                                               Citric acid            3.6    g                                               Water                  516    g                                               ______________________________________                                         Sensory test: 10 panelists                                               

The number of panelists giving the correct answer (discrimination)=4 (nosignificant difference)

EXAMPLE 20 (Acidulant)

A thick-walled cooking pan was charged with granulated sugar, corn syrupand water and heated over a slow fire. As the contents began to boil,the lid was placed in position and the pan was further heated. When theliquid temperature had reached 150° C., an aqueous solution of theacidulant composed of tartaric acid and gluconic acid was added and thefire was extinguished. The contents were transferred to a vat coatedwith salad oil and with the syrup being retained with a spatula, thesyrup was allowed to cool uniformly. When the syrup had cooled, it wascut to size and rounded up to provide a candy. As a comparison example,a candy was similarly manufactured using tartaric acid as acidulant tothe same sourness as the Example. No difference was found between thetwo candies in taste or testure.

    ______________________________________                                        Formulation of Example                                                        Granulated sugar       65     g                                               Powdered corn syrup    4.3    g                                               Tartaric acid          0.66   g                                               50% Gluconic acid      4.14   g                                               Water                  26.2   g                                               Formulation of Comparison Example                                             Granulated sugar       65     g                                               Powdered corn syrup    4.3    g                                               Tartaric acid          1.1    g                                               Water                  28.3   g                                               ______________________________________                                    

EXAMPLE 21 (Acidulant)

A flavored vinegar prepared by mixing vinegar with gluconic acid, commonsalt and granulated sugar was added to cooked rice prepared in theconventional manner and after uniform blending, the rice was allowed tocool to provide a sushi rice stock for chirashi-zushi. As a comparisonexample, a sushi rice stock was similarly prepared using vinegar asacidulant to the same degree of sourness. The two sushi rice stocksorganoleptically showed no significant difference in taste, flavor andtexture.

    ______________________________________                                        Formulation of Example                                                        Cooked rice            300    g                                               Flavored vinegar:                                                             Vinegar                16     g                                               50% Gluconic acid      1.44   g                                               solution                                                                      Granulated sugar       5      g                                               Common salt            1.6    g                                               Formulation of Comparison Example                                             Cooked rice            300    g                                               Flavored vinegar:                                                             Vinegar                20     g                                               Granulated sugar       5      g                                               Common salt            1.6    g                                               ______________________________________                                    

TEST EXAMPLE 8 Effect of Calcium Gluconate on Human Stool Flora

Calcium gluconate, as an example of this invention, was administered toman and its effect on intestinal flora was evaluated.

Six healthy male adult volunteers (aged 28˜56) were instructed to ingestcalcium gluconate powder in a daily dose of 1.7 g once (after lunch)daily and the stool flora was determined at week 1 before intake, on theday of beginning of intake, at weeks 1 and 2 of intake, and weeks 1 and2 of washout after intake by the same method as used in Test Example 3.The results are set forth in Table 22.

                  TABLE 22                                                        ______________________________________                                                   Logarithm of the viable count in each                                         gram of feces*                                                                The FIG. in parentheses denotes the                                           percentage (%) of total count*                                     Microorganism                                                                              Week 1 before intake                                                                         Day 0 of intake                                   ______________________________________                                        Bifidobacterium                                                                            9.5 ± 0.8   9.7 ± 0.7                                                   (17.7 ± 18.3)                                                                             (19.3 ± 19.3)                                  Bacteroidaceae                                                                             10.3 ± 0.4  10.3 ± 0.2                                                  (45.0 ± 27.1)                                                                             (46.9 ± 21.5)                                  Enbacterium  10.0 ± 0.2  10.1 ± 0.2                                                  (25.1 ± 11.8)                                                                             (22.8 ± 7.2)                                   C. perfringens                                                                             6.7 ± 0.4   3.5 ± 0.5                                      (Welch's bacillus)                                                            Enterobacteriaceae                                                                         8.4 ± 1.1   8.3 ± 1.2                                      (inclusive of coliform                                                        bacteria)                                                                     Total viable count                                                                         10.7 ± 0.2  10.7 ± 0.1                                                  (100)          (100)                                             ______________________________________                                                   Logarithm of the viable count in each                                         gram of feces*                                                                The FIG. in parentheses denotes the                                           percentage (%) of total count*                                     Microorganism                                                                              Week 1 of intake                                                                             Week 2 of intake                                  ______________________________________                                        Bifidobacterium                                                                            10.1 ± 0.3  9.9 ± 0.6                                                   (35.8 ± 24.0)                                                                             (30.1 ± 26.9)                                  Bacteroidaceae                                                                             10.1 ± 0.6  10.3 ± 0.4                                                  (35.2 ± 24.5)                                                                             (43.6 ± 23.6)                                  Eubacterium  10.0 ± 0.2  10.0 ± 0.3                                                  (22.9 ± 5.3)                                                                              (19.4 ± 6.9)                                   C. perfringens                                                                             4.0 ± 0.8   4.7 ± 1.4                                      (Welch's bacillus)                                                            Enterobacteriaceae                                                                         8.4 ± 1.0   8.4 ± 1.1                                      (inclusive of coliform                                                        bacteria)                                                                     Total viable count                                                                         10.7 ± 0.3  10.8 ± 0.2                                                  (100)          (100)                                             ______________________________________                                                   Logarithm of the viable count in each                                         gram of feces*                                                                The FIG. in parenthesis denotes the                                           percentage (%) of total count*                                     Microorganism                                                                              Week 1 of washout                                                                            Week 2 of washout                                 ______________________________________                                        Bifidobacterium                                                                            9.7 ± 0.7   9.8 ± 0.8                                                   (32.5 ± 26.2)                                                                             (21.2 ± 18.9)                                  Bacteroidaceae                                                                             10.0 ± 0.8  10.4 ± 0.2                                                  (38.9 ± 28.5)                                                                             (47.1 ± 17.6)                                  Enbacterium  10.0 ± 0.2  10.1 ± 0.2                                                  (27.5 ± 5.9)                                                                              (25.6 ± 13.6)                                  C. perfringens                                                                             3.0 ± 0.6   3.9 ± 2.1                                      (Welch's bacillus)                                                            Enterobacteriaceae                                                                         8.1 ± 1.1   8.2 ± 1.2                                      (inclusive of coliform                                                        bacteria)                                                                     Total viable count                                                                         10.6 ± 0.3  10.8 ± 0.2                                                  (100)          (100)                                             ______________________________________                                         *Mean ± SD                                                            

It is apparent from Table 22 that calcium gluconate increased theintestinal population of bifidobacteria as compared with thepre-ingestion baseline.

Industrial Applicability

The bifidobacterium growth promotant of this invention provides aselective growth promoting effect on bifidobacteria and has excellentcharacteristics as a bifidus factor. Therefore, the bifidobacteriumgrowth promotant of this invention can be used as it is or as added tovarious kinds of foods and beverages to provide functional foods anddrinks and is, hence, of great value from the standpoint of healthpromotion.

We claim:
 1. A composition comprising a bifidobacterium growth promotingamount of an agent selected from the group consisting of gluconic acid,a nontoxic salt of gluconic acid and glucono-δ-lactone.
 2. A functionalsoybean curd comprising not less than 0.5% of a bifidobacterium growthpromotant selected from the group consisting of gluconic acid, anontoxic salt of gluconic acid and glucono-δ-lactone.
 3. A method ofpromoting the growth of bifidobacteria in a patient in need thereofwhich comprises:administering a bifidobacterium growth promoting amountof an agent selected from the group consisting of gluconic acid, anontoxic salt of gluconic acid and glucono-δ-lactone.
 4. The method ofclaim 3, comprising administering at least 1.7 g of said agent per day.5. The method of claim 4, comprising administering at least 9 g of saidagent per day.
 6. A method of controlling intestinal function in apatient in need thereof which comprises:administering a bifidobacteriumgrowth promoting amount of an agent selected from the group consistingof gluconic acid, a nontoxic salt of gluconic acid andglucono-δ-lactone.
 7. The method of claim 6, comprising administering atleast 1.7 g of said agent per day.
 8. The method of claim 7, comprisingadministering at least 9 g of said agent per day.
 9. A method ofattenuating the stool odor in a patient in need thereof whichcomprises:administering a bifidobacterium growth promoting amount of anagent selected from the group consisting of gluconic acid, a nontoxicsalt of gluconic acid and glucono-δ-lactone.
 10. The method of claim 9,comprising administering at least 1.7 g of said agent per day.
 11. Themethod of claim 10, comprising administering at least 9 g of said agentper day.
 12. A method preventing or treating diarrhea in a patient inneed thereof which comprises:administering a bifidobacterium growthpromoting amount of an agent selected from the group consisting ofgluconic acid, a nontoxic salt of gluconic acid and glucono-δ-lactone.13. The method of claim 12, comprising administering at least 1.7 g ofsaid agent per day.
 14. The method of claim 13, comprising administeringat least 9 g of said agent per day.
 15. A method of inhibitingintestinal putrefactive fermentation in a patient in need thereof whichcomprises:administering a bifidobacterium growth promoting amount of anagent selected from the group consisting of gluconic acid, a nontoxicsalt of gluconic acid and glucono-δ-lactone.
 16. The method of claim 15,comprising administering at least 1.7 g of said agent per day.
 17. Themethod of claim 16, comprising administering at least 9 g of said agentper day.
 18. A method of improving stool properties in a patient in needthereof which comprises:administering a bifidobacterium growth promotingamount of an agent selected from the group consisting of gluconic acid,a nontoxic salt of gluconic acid and glucono-δ-lactone.
 19. The methodof claim 18, comprising administering at least 1.7 g of said agent perday.
 20. The method of claim 19, comprising administering at least 9 gof said agent per day.
 21. A method of relieving constipation in apatient in need thereof which comprises:administering a bifidobacteriumgrowth promoting amount of an agent selected from the group consistingof gluconic acid, a nontoxic salt of gluconic acid andglucono-δ-lactone.
 22. The method of claim 21, comprising administeringat least 1.7 g of said agent per day.
 23. The method of claim 22,comprising administering at least 9 g of said agent per day.
 24. Amethod of inhibiting growth of clostridia in a patient in need thereofwhich comprises:administering a bifidobacterium growth promoting amountof an agent selected from the group consisting of gluconic acid, anontoxic salt of gluconic acid and glucono-δ-lactone.
 25. The method ofclaim 24, comprising administering at least 1.7 g of said agent per day.26. The method of claim 25, comprising administering at least 9 g ofsaid agent per day.
 27. The composition of claim 1, further comprisingan additive.
 28. The composition of claim 1, wherein said additive is adietary fiber, excipient, sweetener, nutritional supplement, flavor,binder or lubricant.